ABSTRACT It is now well established that the microbiome is essential to human health. Nearly every month a new study is published linking yet another disease to microbiome dysfunction. This is no longer limited to diseases of the gastrointestinal tract and now includes multiple sclerosis, autism, asthma, Parkinson's, Alzheimer's, and cancer. Culture independent approaches such as sequencing and metabolomics have provided vast amounts of information on the composition and function of the microbiome as a whole. However, in order to enable therapeutic interventions, further understanding of individual microorganisms, their mechanisms and their genes is needed. In this proposal we aim to develop a genome editing tool kit for an important subset of the human microbiota - the genus Lactobacillus. This tool kit will enable researchers to modulate gene expression or abrogate gene function and allow for mechanistic studies of the function of specific genes as well as development of engineered microbiome therapeutics. The phase I proposal will focus on demonstrating the feasibility and increasing the efficiency of the first step in the editing workflow - introduction of a chromosomal edit to a subpopulation of cells using ssDNA recombineering. Utilizing four Lactobacillus species in Phase I, this toolkit will give researchers vital access points into diverse microbiome environments in four distinct geographies on the human body: the stomach, small intestine, large intestine and the female reproductive tract. Phase II studies will focus on optimization of a broad host range vector for the second step of the workflow - Cas9 facilitated selection as well as expansion to additional Lactobacillus species. Moreover the use of the tool kit will be extended to integration or deletion of larger DNA fragments providing a foundation for the development of engineered microbiome therapeutics. When commercialized this tool kit will contain vectors, electroporation reagents, media, strains and design tools that encompass the entire genome editing workflow. These foundational tools will propel further microbiome discoveries and enable the development of novel microbiome interventions.